Hox transcription factors are expressed in unique patterns along the anterior-posterior axis to regulate diverse morphogenetic processes. Members of this protein family bind DNA and regulate gene expression by forming protein complexes with the Hox cofactors, Extradenticle (Exd) and Homothorax (Hth). This finding has led to the hypothesis that Hox proteins select for distinct developmental pathways by controlling the expression of unique combinations of target genes. Interestingly, however, many of the different Hox protein complexes interact with identical or overlapping DNA sequences. Thus, how Hox transcription factors can bind highly related sequences in vitro, and yet direct different developmental pathways in vivo remains unclear. Recently, we, in collaboration with M. Affolter's laboratory (Basel, Switzerland), have characterized a 48 base pair enhancer element (1ab48/95) within the labial (lab) homeobox gene that contains binding sites for Lab, Exd, and Hth. These three transcription factor proteins form a complex with 1ab48/95 in vitro, and each of these binding sites is required to confer gut-specific gene expression in vivo. However, these factors are not sufficient to drive expression of 1ab48/95, as no activity is detected within regions of the head ectoderm, where all three proteins are also co-expressed. These results suggest that additional cofactors are required for the proper spatial expression pattern of the 1ab48/95 enhancer. In this proposal our goals are to: 1) identify the critical nucleotide sequences within 1ab48/95 required for enhancer activity, 2) identify additional proteins that interact with Lab/Exd/Hth and the 1ab48/95 DNA element, and determine their role in the activation of 1ab48/95, and 3) establish an in vivo system to test the ability of additional Hox transcription factors to regulate sequence elements within the endoderm. Taken together, we are optimistic that the characterization of Hox gene activation within the endoderm will serve as a model for how a developmentally-regulated DNA element achieves specificity in vivo.